Subject(s)
Cataract/complications , Lens Capsule, Crystalline/pathology , Lens Implantation, Intraocular , Phacoemulsification , Adult , Cataract/genetics , Child , Female , Humans , Male , Middle Aged , Pedigree , Visual Acuity , VitrectomyABSTRACT
Apoptosis of lens epithelial cells (LECs) is implicated in the pathogenesis of several types of cataract formation. The high intracellular levels of polyol induce histological change in the LECs, which is considered the earliest event in sugar cataractogenesis. This study was designed to investigate whether high galactose exposure induces apoptosis in LECs during the development of sugar cataract. The effect of an aldose reductase inhibitor, SNK-860, was also examined. We induced sugar cataract in Sprague-Dawley rats by feeding them a 50% galactose-containing diet with or without SNK-860. The percentage of LECs undergoing apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL) method, and DNA fragmentation analyses were performed. Galactitol levels in the lens epithelium were quantified by gas chromatography. The number of TUNEL-positive cells gradually increased throughout the period of galactose exposure, up to 5 days. DNA fragmentation analysis in LECs of rats fed a galactose-rich diet demonstrated an apparent ladder pattern. SNK-860 reduced the percentage of TUNEL-positive cells, the amount of intracellular galactitol, and the levels of DNA laddering. To explore the mechanism of the apoptotic process, the expression of p53, a potent mediator of apoptosis, was examined. Based on Western blot and real-time reverse transcription-polymerase chain reaction results, the amount of p53-expression increased at both the protein and mRNA levels after galactose exposure, and the increase in p53-expression was inhibited by SNK-860. Based on these results, we concluded that apoptosis occurs in rat lens epithelial cells following galactose exposure. Furthermore, the reduction of apoptosis by aldose reductase inhibitor suggests that this apoptosis is associated with the accumulation of sugar alcohols. It is probable that the mechanism of apoptosis during sugar cataract formation involves the increased expression of p53.
Subject(s)
Cataract/pathology , Epithelial Cells/pathology , Imidazolidines , Lens, Crystalline/pathology , Aldehyde Reductase/antagonists & inhibitors , Animals , Apoptosis , Blotting, Western/methods , Cataract/metabolism , DNA Fragmentation , Epithelial Cells/metabolism , Galactitol/analysis , Galactose , Genes, p53 , Imidazoles/pharmacology , In Situ Nick-End Labeling , Lens, Crystalline/metabolism , Male , Models, Animal , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Protein p53/analysisABSTRACT
It has been demonstrated that exquisite regulation of the cell cycle between the activation and inhibition is crucial to maintain the transparency of the ocular lens. While it is generally recognized that the sugar cataract is accompanied by the enhanced proliferation of lens epithelial cells (LECs), it is unclear whether or not an inhibitory mechanism against the lens proliferation is involved, except for TGF-beta. In this study, the authors demonstrated the enhanced expression of p21(WAF-1/CIP-1), a potent inhibitor against cell cycle progression, and its specific temporal and regional expression profiles in the LECs during the development of sugar cataract. Sugar cataract was induced in 6-week-old male Sprague-Dawley rats by feeding them on a 50% galactose-rich diet, and then the expression patterns of p21(WAF-1/CIP-1) mRNA and protein with the advance of the sugar cataract were studied. Western blot analyses showed that p21(WAF-1/CIP-1) expression increased throughout the period of galactose exposure, up to 21 days. Also, a gradual increase in the number of p21(WAF-1/CIP-1) positive cells was observed immunohistochemically in the course of the galactose exposure. Interestingly, p21(WAF-1/CIP-1) was significantly expressed in the multi-layered epithelium, which was observed typically in the advanced cataract. Proliferating cell nuclear antigen (PCNA), an indicator of cell proliferation, was also positive in the most multi-layered epithelial cells. In addition, transient expression of PCNA mRNA and its protein was noticed throughout the lens epithelium in the course of the sugar cataract development. Prior to the elevation of p21(WAF-1/CIP-1) mRNA expression, PCNA mRNA expression increased greatly and reached a peak according to the semiquantitative analyses using either the real time reverse transcription-polymerase chain reaction (RT-PCR) or the Southern blot analyses. Based on these observations, it is possible that p21(WAF-1/CIP-1) is elevated and exerts its inhibitory action against the proliferating epithelial cells during the development of the sugar cataract.
